Abstract: The NIH created the Director's New Innovator Award to ""support exceptionally creative new investiga- tors who propose highly innovative projects that have the potential for unusually high impact."" I have been working for more than ten years on new and innovative approaches for manipulating vertebrate genomes. Having the ability to manipulate specific genes in laboratory model systems has been the gold standard of genetic research for many years, but the most useful strategies for specifically knocking out (disrupting) or knocking in (adding) specific genes precisely into the genome has been limited to mice. However, laboratory mice are not always the best model system for understanding human physiology and disease. If we can expand these genome engineering approaches to other species it will have a tremendous impact on human disease research because we will finally be able to study specific genes in the most relevant animal models. Recently, my colleagues and I were the first in the world to target and knock out genes in the laboratory rat, the most widely studied research animal model by physiologists, behavioralists, neuroscientists, nutritionalists, and by researchers in- terested in cardiovascular and kidney diseases. This technological achievement was named by The Scientist as one of the 'Top 10 Innovations'in 2009. By applying a Zinc-Finger Nuclease (ZFN) technology to the rat embryo, generating knockout animal models for specific genes becomes very rapid because it does not require the labor of engineering stem cells and then turning those stem cells into a whole animal. We have been very successful with this approach and have now knocked out 57 genes related to hypertension and renal failure in humans in the past year - something that was not possible even 2 years ago. Using ZFNs for gene knockout, however, is just the beginning of what can be done with this technology. Specifically, we now know that gene knockin approaches in the rat embryo are now possible , but much remains to be explored. We propose to understand the capabilities and limitations of ZFNs of knockin engineering to enable rapid and reproducible methods for addressing hypotheses related to genes involved in any vertebrate model system for virtually any disease or developmental process. For us, the model system is the rat and the disease we study is hypertension, but the innovative approaches developed by this proposal will serve as a blueprint for any model system where embryos can be isolated. Specifically, we will use ZFN knockin technology to enable conditional mutagenesis and whole gene replacement approaches in the rat to develop new and valuable models to address hypotheses related to hypertension and evaluate new anti- hypertensive therapies and make an impact on the estimated 28% of Americans and more than one billion people world-wide affected by this disease. Public Health Relevance: The key to developing effective targeted therapies for the treatment of human disease such is the understanding of how genes, cells and tissues are involved in specific disease processes. Genetic engineering in mice to understand these roles has been the gold standard for many years, but we have developed new approaches which are potentially applicable to any research animal model, including rats. The proposed studies aim to establish highly innovative and widely applicable approaches for studying specific genes in cells and tissues in any model system and to develop better animal models for testing therapies for diseases like hypertension.